In the early stages of cell differentiation, certain genes switch off and others switch on. In the ML-1 human myeloblastic leukemia cell line, we have shown that c-myb switches off early in induction with 12-0-tetradecanoylphorbol-13-acetate (TPA). Our goal now is to identify and characterize genes that switch on early in induction. We are making considerable progress towards this goal: By optimizing conditions, we shortened the induction period to 1-4 hours (from 1-3 days). Using differential screening, we isolated two CDNA clones which increase in expression (>8fold) during induction with TPA. Expression increases very early (at 1-3 hours), which is prior to the appearance of early differentiation markers (at -0.5-1 day) and differentiated morphology (at -3 days). Thus, these cDNAs represent "early-induction" genes in that they are expressed during commitment to differentiation and before the appearance of maturing phenotype. Interestingly, initial sequence data from one of these cDNAs reveals no extensive areas of homology with other known genes. We now aim to further explore the role of the early-induction genes in differentiation, proliferation, and leukemia (Aim 1). We will monitor the time course of expression with a variety of inducers (versus non-inducers). We will monitor expression under other conditions of changing proliferation and will look for alterations in leukemic cells at various differentiation stages.' These studies will highlight genes likely to play an important role in early-induction and/or leukemogenesis. We also aim to molecularly characterize the early-induction genes (Aim 2). We are determining cDNA sequence and will examine the regulation of transcription (nuclear run-on assays). We will assess how gene expression affects differentiation (using antisense RNA techniques and gene transfer). Finally, we aim to identify additional early-induction genes (Aim 3), using more sensitive screening techniques. This will involve enriching probes for sequences increased by more than one inducer and depleting them of sequences present in nondifferentiating cells. These studies will advance us towards our long-term goal of understanding the molecular events that underlie the triggering of differentiation in ML-1.